Various pulmonary and respiratory diseases, such as pneumonia, bronchitis, and cystic fibrosis, are often accompanied by viscous secretions in respiratory airways of the patient. Obstruction of the airways by such secretions can cause respiratory distress, and in some cases, can lead to respiratory failure and death.
Investigators have compared the Na.sup.+ and Cl.sup.- permeability of normal epithelia and epithelia in patients suffering from such diseases described above. [See, e.g., Welsh et al., J. Clin. Invest., 80:1523-1526 (1987)]. Measurements of short-circuit current ("I.sub.sc ") have shown that normal human airway epithelia are predominantly Na.sup.+ absorbing under baseline conditions while Cl.sup.- secretion is relatively small or absent [Smith et al., J. Clin. Invest., 91:1590-1597 (1993)]. Baseline Na.sup.+ absorption, however, is elevated approximately two-fold in cystic fibrosis ("CF") patients [Boucher et al., J. Clin. Invest., 78:1245-1252 (1986)].
Recently, a cAMP-dependent Cl.sup.- channel has been identified to be defective in CF patients. This channel, referred to as CF transmembrane regulator ("CFTR"), has been suggested to play an important role in normal Na.sup.+ transport [Stutts et al., Science, 269:847-850 (1995)]. Despite the presence of several Cl.sup.- channels being present in the apical membrane of airway epithelial cells, it is believed that the CFTR provides the major Cl.sup.- conductance pathway in these cells and that this channel is close to maximally activated under baseline conditions [Shen et al., Am. J. Phys., 269:L561-L566 (1995)]. In the absence of significant Cl.sup.- efflux, the increased basal Na.sup.+ influx (and associated fluid absorption) observed in CF airway epithelia has been suggested to contribute to the formation of the abnormally hydrated, viscous mucous secretions which occlude the airways in CF disease [Jiang et al., Science, 262:424-427 (1993)].
Certain pharmacological approaches to rectify the abnormal fluid absorption in CF patients have been described. For instance, some investigators have described stimulating epithelial Cl.sup.- secretion using agents such as uracil 5' triphosphate ("UTP") to raise intracellular calcium ion levels and attenuate Ca.sup.2+ activated Cl.sup.- channels [Knowles et al., N. Engl. J. Med., 325:533-538 (1991)]. Another approach has been to use aerosolized amiloride to block Na.sup.+ absorption [Boucher et al., supra]. The present invention is directed to the use of hepatocyte growth factor ("HGF") to modulate fluid absorption by modulating the expression of Na.sup.+ channels. As described below, the present inventors have surprisingly discovered that HGF produces an effective and prolonged block of amiloride-sensitive Na.sup.+ absorption.
It is recognized in the art that HGF functions as a growth factor for particular tissues and cell types. HGF was identified initially as a mitogen for hepatocytes [Michalopoulos et al., Cancer Res., 44:4414-4419 (1984); Russel et al., J. Cell. Physiol., 119:183-192 (1984); Nakamura et al., Biochem. Biophys. Res. Comm., 122:1450-1459 (1984)]. Nakamura et al., supra, reported the purification of HGF from the serum of partially hepatectomized rats. Subsequently, HGF was purified from rat platelets, and its subunit structure was determined [Nakamura et al., Proc. Natl. Acad. Sci. USA, 83:6489-6493 (1986); Nakamura et al., FEBS Letters, 224:311-316 (1987)]. The purification of human HGF ("huHGF") from human plasma was first described by Gohda et al., J. Clin. Invest., 81:414-419 (1988).
Both rat HGF and huHGF have been molecularly cloned, including the cloning and sequencing of a naturally occurring variant lacking 5 amino acids designated "delta5 HGF" [Miyazawa et al., Biochem. Biophys. Res. Comm., 163:967-973 (1989); Nakamura et al., Nature, 342:440-443 (1989); Seki et al, Biochem. Biophys. Res. Commun., 172:321-327 (1990); Tashiro et al., Proc. Natl. Acad. Sci. USA, 87:3200-3204 (1990); Okajima et al., Eur. J. Biochem., 193:375-381 (1990)].
The mature form of huHGF, corresponding to the major form purified from human serum, is a disulfide linked heterodimer derived by proteolytic cleavage of the human pro-hormone between amino acids R494 and V495. This cleavage process generates a molecule composed of an .alpha.-subunit of 440 amino acids (M.sub.r 69 kDa) and a .beta.-subunit of 234 amino acids (M.sub.r 34 kDa). The nucleotide sequence of the huHGF cDNA reveals that both the .alpha.- and the .beta.-chains are contained in a single open reading frame coding for a pre-pro precursor protein. In the predicted primary structure of mature huHGF, an interchain S-S bridge is formed between Cys 487 of the .alpha.-chain and Cys 604 in the .beta.-chain [see Nakamura et al., Nature, supra]. The N-terminus of the .alpha.-chain is preceded by 54 amino acids, starting with a methionine group. This segment includes a characteristic hydrophobic leader (signal) sequence of 31 residues and the prosequence. The .alpha.-chain starts at amino acid (aa) 55, and contains four kringle domains. The kringle 1 domain extends from about aa 128 to about aa 206, the kringle 2 domain is between about aa 211 and about aa 288, the kringle 3 domain is defined as extending from about aa 303 to about aa 383, and the kringle 4 domain extends from about aa 391 to about aa 464 of the .alpha.-chain.
The definition of the various kringle domains is based on their homology with kringle-like domains of other proteins (such as prothrombin and plasminogen), therefore, the above limits are only approximate. To date, the function of these kringles has not been determined. The .beta.-chain of huHGF shows high homology to the catalytic domain of serine proteases (38% homology to the plasminogen serine protease domain). However, two of the three residues which form the catalytic triad of serine proteases are not conserved in huHGF. Therefore, despite its serine protease-like domain, huHGF appears to have no proteolytic activity, and the precise role of the .beta.-chain remains unknown. HGF contains four putative glycosylation sites, which are located at positions 294 and 402 of the .alpha.-chain and at positions 566 and 653 of the .beta.-chain.
In a portion of cDNA isolated from human leukocytes, in-frame deletion of 15 base pairs was observed. Transient expression of the cDNA sequence in COS-1 cells revealed that the encoded HGF molecule (delta5 HGF) lacking 5 amino acids in the kringle 1 domain was fully functional [Seki et al., supra].
A naturally occurring huHGF variant has been identified which corresponds to an alternative spliced form of the huHGF transcript containing the coding sequences for the N-terminal finger and first two kringle domains of mature huHGF [Chan et al., Science, 254:1382-1385 (1991); Miyazawa et al., Eur. J. Biochem., 197:15-22 (1991)]. This variant, designated HGF/NK2, has been proposed to be a competitive antagonist of mature huHGF.
Comparisons of the amino acid sequence of rat HGF with that of huHGF have revealed that the two sequences are highly conserved and have the same characteristic structural features. The length of the four kringle domains in rat HGF is exactly the same as in huHGF. Furthermore, the cysteine residues are located in exactly the same positions, an indication of similar three-dimensional structures [Okajima et al., supra; Tashiro et al., supra].
HGF and HGF variants are described further in U.S. Pat. Nos. 5,227,158; 5,316,921; and 5,328,837.
The HGF receptor has been identified as the product of the c-Met proto-oncogene [Bottaro et al., Science, 251:802-804 (1991); Naldini et al., Oncogene, 6:501-504 (1991); WO 92/13097 published Aug. 6, 1992; WO 93/15754 published Aug. 19, 1993]. The receptor is usually referred to as "c-Met" or "p190.sup.MET " and typically comprises, in its native form, a 190-kDa heterodimeric (a disulfide-linked 50-kDa .alpha.-chain and a 145-kDa .beta.-chain) membrane-spanning tyrosine kinase protein [Park et al., Proc. Natl. Acad. Sci. USA, 84:6379-6383 (1987)]. Several truncated forms of the c-Met receptor have also been described [WO 92/20792; Prat et al., Mol. Cell. Biol., 11:5954-5962 (1991)].
The binding activity of HGF to its receptor is believed to be conveyed by a functional domain located in the N-terminal portion of the HGF molecule, including the first two kringles [Matsumoto et al., Biochem. Biophys. Res. Commun., 181:691-699 (1991); Hartmann et al., Proc. Natl. Acad. Sci., 89:11574-11578 (1992); Lokker et al., EMBO J., 11:2503-2510 (1992); Lokker and Godowski, J. Biol. Chem., 268:17145-17150 (1993)]. The c-Met protein becomes phosphorylated on tyrosine residues of the 145-kDa .beta.-subunit upon HGF binding.
Various biological activities have been described for HGF and its receptor [see, generally, Chan et al., Hepatocyte Growth Factor-Scatter Factor (HGF-SF) and the C-Met Receptor, Goldberg and Rosen, eds., Birkhauser Verlag-Basel (1993), pp. 67-79]. It has been observed that levels of HGF increase in the plasma of patients with hepatic failure [Gohda et al., supra] and in the plasma [Lindroos et al., Hepatol., 13:743-750 (1991)] or serum [Asami et al., J. Biochem., 109:8-13 (1991)] of animals with experimentally induced liver damage. The kinetics of this response are usually rapid, and precedes the first round of DNA synthesis during liver regeneration. HGF has also been shown to be a mitogen for certain cell types, including melanocytes, renal tubular cells, keratinocytes, certain endothelial cells and cells of epithelial origin [Matsumoto et al., Biochem. Biophys. Res. Commun., 176:45-51 (1991); Igawa et al., Biochem. Biophys. Res. Commun., 174:831-838 (1991); Han et al., Biochem., 30:9768-9780 (1991); Rubin et al., Proc. Natl. Acad. Sci. USA, 88:415-419 (1991)]. Both HGF and the c-Met protooncogene have been postulated to play a role in microglial reactions to CNS injuries [DiRenzo et al., Oncogene, 8:219-222 (1993)].
HGF can also act as a "scatter factor", an activity that promotes the dissociation of epithelial and vascular endothelial cells in vitro [Stoker et al., Nature, 327:239-242 (1987); Weidner et al., J. Cell Biol., 111:2097-2108 (1990); Naldini et al., EMBO J., 10:2867-2878 (1991); Giordano et al., Proc. Natl. Acad. Sci. USA, 90:649-653 (1993)]. Moreover, HGF has recently been described as an epithelial morphogen [Montesano et al., Cell, 67:901-908 (1991)]. Therefore, HGF has been postulated to be important in tumor invasion [Comoglio, Hepatocyte Growth Factor-Scatter Factor (HGF-SF) and the C-Met Receptor, Goldberg and Rosen, eds., Birkhauser Verlag-Basel (1993), pp. 131-165].